Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle that alternates between various species of small mammals and a tick vector. B. burgdorferi has an unusual segmented genome that includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded genes are critical for successful adaptation by the spirochete to the different environments that it encounters during its infectious cycle. We have developed genetic tools that we use to investigate basic aspects of this bacterium's unusual genomic organization, cellular structure and metabolism. We have extended this investigation to an in vivo setting with an experimental system that closely mimics the natural arthropod vector/rodent host infectious cycle. Through an understanding of the basic molecular biology of the organism, we hope to gain insight into the infectious strategy utilized by this significant vector-borne pathogen and thereby facilitate efforts to prevent, diagnose and treat Lyme disease. Environmental changes that accompany tick feeding are sensed by B. burgdorferi and utilized as cues to induce critical switches in gene expression and protein synthesis. This response includes the induction of outer surface protein C (OspC), a hallmark of adaptation to the mammalian environment. We have established that OspC is required by B. burgdorferi to initiate infection of the mammalian host, but is subsequently targeted by the host's acquired immune response and therefore must be down regulated for persistent infection. We previously identified BBD18, a unique plasmid-encoded protein of B. burgdorferi, as a potent repressor of OspC expression. In FY2014 we investigated the in vivo role of BBD18 in an experimental mouse-tick model system (1). We showed that constitutive production of BBD18 by B. burgdorferi does not alter colonization or persistence in ticks, but prevents induction of OspC during tick feeding and blocks infection in mice. However, spirochetes retain infectivity in mice when constitutively producing BBD18 variants with site-directed mutations that eliminate OspC repression. Importantly, we showed that spirochetes lacking BBD18 can persistently infect mice, but are not acquired by larval ticks. Finally, we demonstrated that a previously described OspC operator sequence is not required for repression of OspC by BBD18 in B. burgdorferi. We conclude that BBD18 represses OspC as part of the adaptive response of B. burgdorferi, most likely during spirochete acquisition by larval ticks, and does not represent the OspC-specific repressor that is needed for persistent infection of the mammalian host. As described above, B. burgdorferi adapts in order to survive in the dissimilar environments encountered during the infectious cycle. One way this is accomplished is through the use of alternative sigma factors to direct transcription of specific genes. RpoS, one of only three sigma factors in B. burgdorferi, controls expression of genes required during tick-transmission and infection of the mammalian host. In FY2014 we established a role for BBD18 in the regulation of the virulence-associated sigma factor RpoS (2). Constitutive expression of BBD18 repressed transcription of RpoS-dependent genes to levels equivalent to those observed in an RpoS mutant, but did not diminish the amount of RpoS transcript, indicating post-transcriptional regulation of RpoS by BBD18. Interestingly, BBD18-mediated repression of RpoS is independent of both the RpoS promoter and the 5' untranslated region, suggesting a mechanism of protein destabilization rather than translational control. We conclude that BBD18 is not the OspC-specific repressor required for persistent mammalian infection as we initially proposed, but rather serves as a master regulator during spirochete acquisition by feeding ticks, to switch off RpoS-dependent genes required in the vertebrate host and switch on a new set of genes required exclusively in the tick vector. The population dynamics of B. burgdorferi throughout its natural infectious cycle are not well understood. In FY2014 we addressed this topic by assessing the colonization, dissemination and persistence of B. burgdorferi within and between the disparate mammalian and tick environments (3). To follow bacterial populations during infection, we generated seven isogenic but distinguishable B. burgdorferi clones, each with a unique sequence tag. These tags resulted in no phenotypic changes relative to wild type organisms, yet permitted highly sensitive and specific detection of individual clones by PCR. We followed the composition of the spirochete population throughout an experimental infectious cycle that was initiated with a mixed inoculum of all clones. We observed heterogeneity in the spirochete population disseminating within mice at very early time points, but all clones displayed the ability to colonize most mouse tissues by 3 weeks of infection. The complexity of clones subsequently declined as murine infection persisted. Larval ticks typically acquired a reduced and variable number of clones relative to what was present in infected mice at the time of tick feeding, and maintained the same spirochete population through the molt to nymphs. However, only a random subset of infectious spirochetes was transmitted to nave mice when these ticks next fed. Our results clearly demonstrate that that both acquisition and transmission of B. burgdorferi by feeding ticks represent bottlenecks that stochastically limit the complexity of the spirochete population. The experimental system that we have developed can be used to further explore the forces that shape the population of this vector-borne bacterial pathogen throughout its infectious cycle. 1. Hayes BM, Dulebohn DP, Sarkar A, Tilly K, Bestor A, Ambroggio X, Rosa PA (2014) Regulatory protein BBD18 of the lyme disease spirochete: essential role during tick acquisition? MBio 5:e01017-14 2. Dulebohn DP, Hayes BM, Rosa PA (2014) Global repression of host-associated genes of the Lyme disease spirochete through post-transcriptional modulation of the alternative sigma factor RpoS. PLoS One 9:e93141 3. Rego RO, Bestor A, Stefka J, Rosa PA (2014) Population bottlenecks during the infectious cycle of the Lyme disease spirochete Borrelia burgdorferi. PLoS One 9:e101009